VLPs as Vaccines.
The growth of recombinant DNA technology in recent years has led to the introduction of vaccines in which an immunogenic protein has been identified, cloned and expressed in a suitable host to obtain sufficient quantities of protein to allow effective protective immunization in both animals and humans. Many of the most effective vaccines are based on the potent ability of virion surfaces to elicit neutralizing antibodies. These include licensed killed or attenuated virus vaccines, such as polio, influenza and rabies, which effectively induce protective antibody responses. More recently, subunit vaccines based upon self-assemblages of the structural proteins of human papillomavirus (HPV) and hepatitis B virus (HBV) have been approved by the Food and Drug Administration. The subunits are expressed in a suitable host and then self-assemble into particles that structurally resemble authentic viruses, but are noninfectious because they lack the viral genome. These so-called virus-like particles (VLPs) in general are highly immunogenic, because the structural proteins of which they are comprised are present in multiple copies in each individual particle. This high density of antigen presentation makes these particles especially effective at provoking a robust antibody response. The HBV and HPV vaccines are based on VLPs assembled from the structural proteins of the respective viruses themselves, but VLPs can also be utilized as scaffolds for the high-density display of heterologous epitopes. Since VLPs in general represent highly repetitive and, therefore, highly immunogenic structures, they may be derived from any number of different virus types. The present method is directed toward utilizing the VLPs of RNA bacteriophages (especially MS2 and PP7) both for immunogenic display and for epitope discovery by a method analogous to phage display [1, 2].
RNA Bacteriophages.
The single-strand RNA bacteriophages are a group of viruses found widely distributed in nature. Several have been characterized in great detail in terms of genome sequence, molecular biology, and capsid structure and assembly. MS2 is perhaps the best-studied member of the group and has been the focus of most of the work performed in the inventors's laboratories, although recent work also exploits a related phage called PP7. MS2 has a 3569-nucleotide single-strand RNA genome that encodes only four proteins: maturase, coat, lysis and replicase. The viral particle is comprised of 180 coat polypeptides, one molecule of maturase, and one copy of the RNA genome. Since the coat protein itself is entirely responsible for formation of the icosahedral shell, the MS2 VLP can be produced from plasmids as the product of a single gene. Thus, in comparison to the other phages used for peptide display, RNA VLPs are strikingly simple. The engineering of MS2 and PP7 VLPs for peptide display and affinity selection has been presented recently by these inventors [1, 2] and is also described later in this document.
Epitope Identification by Conventional Phage Display.
Phage display is one of several technologies that make possible the presentation of large libraries of random amino acid sequences with the purpose of selecting from them peptides with certain specific functions (e.g. the ability to bind a specific antibody). The most commonly used phage display method is based on the filamentous phages (e.g. M13). The basic idea is to create recombinant bacteriophage genomes containing a library of randomized sequences genetically fused in the phage's DNA genome to one of the viral structural proteins. When such recombinants are transfected into bacteria, each produces a virus particle that displays a particular peptide on its surface and packages the same recombinant genome that encodes the peptide. This establishes the linkage of genotype and phenotype essential to the method. Arbitrary functions (e.g. the binding of a receptor, immunogenicity) can be selected from complex libraries of peptide-displaying phages by the use of affinity-selection followed by amplification of the selectants by growth in E. coli. In a vast library of peptide-displaying phages, the tiny minority able to bind a particular receptor (e.g. a monoclonal antibody) can be affinity purified and then amplified by propagation in E. coli. Usually several iterative rounds of selection and amplification are sufficient to yield a relatively simple population from which individual phages displaying peptides with the desired activity can be cloned and then characterized. When the selecting molecule is an antibody, the peptides thus identified represent epitopes recognized by the antibody, and, under appropriate conditions, may be able to evoke in an immunized patient or animal an antibody response specific for the epitope in its native antigen.
However, there are disadvantages to filamentous phage display. Most importantly, a quirk of filamentous phage molecular biology often makes it difficult or impossible to display peptides at the high densities necessary for really potent immunogenicity. This means that although peptide epitopes may be identified by affinity selection, to be useful as immunogens (i.e. vaccines) they must usually be synthesized chemically and then conjugated to a more immunogenic carrier. Unfortunately, the peptide frequently loses activity when thus divorced from the structural context in which it resided during its affinity selection and optimization. The RNA phage VLP display system described here, on the other hand, creates the ability to conduct affinity selection and immungenic epitope presentation on a single platform. This is a consequence of combining high-density peptide epitope display with an affinity-selection capability, and means that the structural constraints present during the epitope's affinity selection can be maintained during the immunization process, increasing the likelihood that the epitope will retain the structure necessary to elicit the desired antibody response.
Overview of the RNA Phage VLP Display Method.
The inventors previously described a technology for peptide display and affinity selection based on the VLPs of RNA bacteriophages, including MS2 and PP7 [1, 2], and explain it briefly here. Development of the VLP display method required that two preconditions be satisfied: First it was necessary to identify a form of the RNA phage coat protein, and a site within it that tolerated insertion of foreign peptides without disruption of its ability to properly fold and assemble into a VLP. The AB-loop on the surface of coat protein was chosen as the site for peptide insertion. Peptides inserted here are prominently displayed on the surface of the VLP. Unfortunately, the wild-type form of coat protein is highly intolerant of peptide insertions in the AB-loop, with the vast majority (usually >98%) leading to folding failures. Coat protein normally folds as a dimer, ninety of which assemble into the icosahedral VLP. The inventors engineered a novel form of coat protein to stabilize it and to render it more tolerant of AB-loop insertions. To do so they took advantage of the proximity of the N- and C-termini of the two identical polypeptide chains in the dimer (FIG. 1). By duplicating the coat protein coding sequence and then fusing the two copies into a single reading frame, the inventors produced a so-called single-chain dimer. This form of the protein is dramatically more stable thermodynamically and its folding is vastly more tolerant of peptides inserted into the AB-loop of the downstream copy of the single-chain dimer [1, 2]. The resulting VLPs display one peptide per dimer, or ninety peptides per VLP. The second precondition for a peptide display/affinity-selection capability is the linkage of phenotype to genotype, as it is essential to provide a means to amplify affinity-selected sequences. This requirement was satisfied when the inventors showed that RNA phage VLPs encapsidate the messenger-RNA that directs their synthesis [1, 2]. This means that the sequences of affinity-selected peptide-VLPs can be amplified by reverse transcription and polymerase chain reaction. When the selection target is a monclonal antibody, the resulting affinity selected VLPs represent vaccine candidates for elicitating in animals or patients of antibodies whose activities mimic that of the selecting antibody.
Considerations Related to Peptide Display Valency and its Control.
This application describes plasmid vectors that facilitate the construction of complex random sequence and antigen fragment libraries on RNA phage VLPs and the affinity selection from such libraries of peptides that bind specific monoclonal antibodies (or other arbitrary receptors). Note that as originally described, the RNA phage VLP display technology presents 90 peptides on each VLP since the peptide is inserted in one AB-loop of a single-chain dimer, and 90 dimers make up the VLP [1, 2]. The multivalency of these particles is desirable for most applications of the MS2 VLP. For example, the high immunogenicity of the particle is related to the high density of the peptides displayed, and is thus a valued property in a vaccine. However, during the affinity selection process, multivalency makes it difficult to distinguish particles that display peptides with intrinsic high binding affinity for the selection target from those that bind tightly only because of multiple simultaneous weak interactions. This “avidity vs. affinity” dilemma is a well-documented complication in the selection of high affinity peptide ligands using filamentous phage display [3-5]. The present invention addresses this issue in the VLP display system by introducing a means of adjusting average peptide display valency levels over a wide range, i.e., from fewer than one to as many as ninety per particle. This makes it possible to alter the density of peptide display during the affinity-selection process. Selection is conducted in several rounds, with the first round typically conducted using multivalent display, thus obtaining a relatively complex population including all peptides having some minimal affinity for the target. In subsequent rounds the peptide display valency can be reduced, thus increasing the selection stringency, and resulting in the isolation of peptides with higher affinity for the antibody target, and better molecular mimics of the preferred epitope.